The experiments outlined in this proposal are designed to provide a deeper understanding of the mechanisms by which viruses select and package multipartite genomes into a single particle. During the previous funding period we have focused our attention on the bipartite, positive-strand RNA nodavirus Flock House virus (FHV), which represents a relatively simple system to address this issue. By using a combination of molecular and cell biological approaches we discovered that specific genome packaging relies heavily on defined subcellular location of the viral coat protein and the genomic RNAs. Specifically, our data suggest that the two genomic segments are packaged independently and in different cellular micro-environments. In addition, we have evidence that both ER and mitochondria play a critical role in this process. Our data have been summarized in an updated model for nodaviral RNA packaging and we intend to confirm and further refine this model in the next funding period. We believe that the experiments outlined in this proposal will not only illuminate an important aspect of molecular virology but also reveal new facets of cell biology. In aim 1, we will determine the cellular location of viral RNA2, the message of FHV coat protein, and map its site of translation. In aim 2, we will determine how coat protein trafficks from the ER to mitochondria, the site of viral RNA synthesis, and whether cellular factors are involved in this process. In aim 3, we will identify determinants of specific FHV genome packaging, specifically whether genome segments are packaged co-translationally or co- transcriptionally and how these mechanisms determine formation of reassortants during mixed infections. PUBLIC HEALTH RELEVANCE: Assembly of virus particles requires coordinated interactions between viral capsid proteins and the viral genome. Some viruses, such as the influenza viruses, package multiple genome segments into a single particle and the mechanisms by which these segments are selected are not understood. We study a small insect virus that packages two genome segments into the same particle and we have discovered that the capsid protein of this virus selects the two genomic RNAs independently and in different locations of the cell. These studies have provided the first clues as to how viruses regulate packaging of segmented genomes. In this proposal we plan to further investigate the molecular details of this mechanism in hopes of providing insights as to how other viruses deal with a similar problem.